Mechanisms of COPII-Dependent Quality Control and ER Export The sorting and transport of biosynthetic cargo from the endoplasmic reticulum (ER) is central to eukaryotic cell physiology. This proposal is focused on the conserved mechanisms and machinery molecules, in particular the COPII vesicular coat proteins, responsible for the controlled export of thousands of distinct cargo proteins from the ER. Newly synthesized proteins are subject to quality control (QC) interrogations that may involve repeated attempts at chaperone-mediated folding, or that target aberrantly folded forms for destruction by ER- associated degradation (ERAD) (1). It is also the case that QC decisions are made at ER exit sites, as the COPII coat and associated machinery proteins actively exclude misfolded cargo from vesicles (2, 3). The mechanisms that control segregation of folded cargo from ER chaperones and misfolded proteins, and their contribution to ER quality control, are not well understood. This research will address central questions of COPII trafficking relevant to cell physiology and pathophysiology. The specific aims of the proposal are to: (Aim 1) carry out a structure-function mapping of COPIImachinery interactions, with which to construct a pseudo- atomic model of the COPII vesicle interior. Building on prior work we will complete the mapping for all 12 major transmembrane protein constituents of COPII vesicles (22). We will identify ER export motifs on the circulating ER-Golgi receptor proteins Erv41/Erv46, Rer1 and Erv29, and determine their crystal structures in complex with COPII protein. Functional relevance will be tested definitively in COPII budding reconstitutions and whole cell experiments; (Aim 2) investigate the mechanism of COPII protein sorting that imposes ER retention, and explore its contribution to QC. We will test the complex interplay of cargo, chaperones and COPII-associated machinery that underlies ER retention (2), exploiting a set of tools developed in the initial phase of research? in particular, a novel series of small molecules that bind COPII protein to reduce the stringency of ER retention. We will investigate the capacity of the retention and retrieval systems, the breakdown of retention in UPR- activated cells, and we will test our working model that chaperone complexes are excluded from vesicles because they are too large to partition into the interstices of a COPII-associated luminal array; and (Aim 3) develop the new idea of COPII cargo uptake by enhanced partitioning as an alternative to the bulk flow model for ER exit. The ER-to-Golgi transport step is essential for eukaryotic cell growth and function, thus the proposed studies have considerable relevance to human cell physiology and to understanding diseases of protein mistrafficking and proteotoxicity.